99

Journal of Physiology (1992), 458, pp. 99-117 With 9 figures Printed in Great Britain

CAFFEINE-INDUCED RELEASE AND REUPTAKE OF Ca2+ BY Ca2+ STORES IN MYOCYTES FROM GUINEA-PIG URINARY BLADDER

BY V. YA. GANITKEVICH AND G. ISENBERG From the Department of Physiology, University of Cologne, Robert-Koch straJ3e 39, D-5000 Koln-41, Germany

(Received 3 January 1992) SUMMARY

1. Voltage-clamped isolated smooth muscle cells from guinea-pig urinary bladder were studied with 3-6 mm extracellular Ca2+ at 36 'C. The fluorescence of the Ca2+_ sensitive dye Indo- 1 was used to monitor the cytosolic calcium concentration ([Ca2+]1) and its changes ([Ca2+]i transient). Fast application of caffeine (10 mM) to the cell was used to release the intracellular Ca2+ from a 'caffeine-sensitive Ca2+ store'. 2. At the holding potential -60 mV, a short (1 s) caffeine application increased [Ca2+]i within less than 1 s from the resting 118 + 22 nm to 1490 + 332 nm. Following the caffeine wash-out, [Ca2+]i fell from this peak to a subresting level of 47 + 12 nM, i.e. an 'undershoot' of [Ca2+]i occurred. Subsequent caffeine-induced [Ca2+]i transients had attenuated peaks suggesting that the caffeine-sensitive Ca2+ store had lost a part of the releasable Ca2+. 3. In the continuous presence of caffeine, [Ca2+]i decayed from its peak to control resting [Ca2+]i values. The wash-out of caffeine following prolonged (10-30 s) treatment also resulted in [Ca2+]i undershoot. Subsequent caffeine-induced [Ca2+]i transients were largely abolished as if the caffeine-sensitive Ca2+ store had lost a large part of releasable Ca2+. During the undershoot, hyperpolarization to - 100 mV did not affect [Ca2+]i. In most cells studied, recovery of [Ca2+]i from the undershoot to the resting level required depolarizations inducing Ca21 influx through L-type Ca2+ channels. 4. Block of plasmalemmal Ca2+-ATPase (PMCa) with extracellular La3` (3 mM) did not modify the decay of the [Ca2+]i transients induced by depolarization or by a 1 s caffeine application suggesting that decay rate of both is not limited by PMCa rate. La3+ abolished the undershoot of [Ca2+]i. In the continuous presence of caffeine, La3+ largely prevented the decay of [Ca2+]i. 5. When the depolarizing steps from -60 to 0 mV (160 ms duration) were applied during the period of [Ca21]i undershoot, the half-time of decay of the corresponding [Ca2+]i transients was up to three times faster than in control. Repetitive depolarizations restored the rate of decay and [Ca2+]i recovered to the resting value. Both processes recovered along a similar time course. 6. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 041 mM) or of 8-Br-cAMP (0 1 mM) did not mimic the above caffeine effects MS 1010

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V. YA. GANITKEVICH AND G. ISENBERG

suggesting that stimulation of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCa) by cAMP-dependent phosphorylation is not the underlying mechanism. A Ca2 , calmodulin-dependent phosphorylation is also unlikely to play a role since the decay rate of the depolarization-induced [Ca2+]i transients increased at reduced [Ca2+]i. 7. It is suggested that the undershoot of [Ca2+]i is due to a reduction of the Ca2+ content in the cell due to the plasmalemmal Ca2+-ATPase that extrudes part of the Ca2+ released by caffeine. The concomitant reduction of [Ca2+] in the SR is thought to stimulate the rate of the SR Ca2+-ATPase and therefore accelerate the decay of the depolarization-induced [Ca2+]i transient. INTRODUCTION

In smooth muscle caffeine is widely used as a tool for studying the Ca2+ release from the sarcoplasmic reticulum (SR) (Deth & Casteels, 1977; Jino, 1989). It has been thought to interact with the mechanism of Ca2+-induced release of Ca2+ (CIRC; Endo, 1988). More recently, this hypothesis was proved at the molecular level. Caffeine activates the reconstituted Ca2+-release channels which were isolated as ryanodine receptors from cardiac (Rousseau & Meissner, 1989; Sitsapesan & Williams, 1990) or vascular SR (Herrmann-Frank, Darling & Meissner, 1991). It is likely that the caffeine activation is the result of a sensitization of the release channel to Ca2+ inducing CIRC (Sitsapesan & Williams, 1990). Originally, the Ca2+ release was quantified by the caffeine-induced force transient, Ca2+ influx being prevented by a Ca2+-free media. Direct measurements of [Ca2+]i are preferable since it has become clear that caffeine produces complex effects on smooth muscle cells (Saida & van Breemen, 1984; Ahn, Karaki & Urakawa, 1988; Ozaki, Kasai, Hori, Sato, Ishihara & Karaki, 1990). The caffeine-induced changes in [Ca2+]i have been used to define the 'caffeine-sensitive Ca2+ store' in skinned multicellular preparations (Iino, 1989). Comparison of the [Ca2+]i changes induced by caffeine and by inositol 1,4,5-trisphosphate (IP3) suggested that the caffeine-sensitive Ca2+ store is smaller and of less physiological importance than the IP3-sensitive Ca2+ store (lino, 1989, 1990a). However, the results from experiments with skinned preparations are difficult to extrapolate to the intact cell. Experimental conditions used (such as buffering of the intracellular [Ca2+]i with 10 mm EGTA) seem to suppress the physiological feedback mechanisms when a small increment in [Ca2+]i produces further CIRC. In the present study, the experiments were performed on single voltage-clamped smooth muscle cells isolated from guinea-pig urinary bladder where Ca2+ fluxes through the Na+-Ca2+ exchanger can be neglected (Ganitkevich & Isenberg, 1991). The caffeine-induced changes in [Ca2+]i were recorded at a membrane potential of -60 mV thereby preventing possible effects of caffeine on membrane potential and on Ca2+ influx through L-type Ca2+ channels. Under these conditions, the caffeineinduced changes in [Ca2+]i were the result of Ca2+ release superimposed on Ca2+ reuptake into SR and Ca2+ extrusion from the cell. A part of this work has been presented to the Physiological Society (Ganitkevich & Isenberg, 1992a).

CAFFEINE-SENSITIVE Ca2+ STORE IN SMOOTH MUSCLE

lot

METHODS

Adult guinea-pigs were killed by cervical dislocation, and then the urinary bladder was removed. The methods of cell isolation, recording of whole-cell membrane currents as well as the measurements of the Indo- 1 fluorescence and calibration of [Ca2+]i were used as described in detail before (Ganitkevich & Isenberg, 1991). Briefly, cells were voltage clamped with patch electrodes (2-4 MQ resistance) and whole-cell membrane currents filtered at 1 kHz were measured with an RK-300 patch-clamp amplifier (Biologic, Echirolles, France). Following the establishment of the whole-cell recording mode, at least 2 min of loading with Indo-1 was allowed before starting the experiment. For microfluospectroscopic measurements the cell was illuminated at 340 nm through an objective (Nikon, 100 x oil immersion) with a 75 W xenon lamp. Emitted light in bands from 395 to 425 nm and 450 to 490 nm was collected and amplified by a pair of photomultipliers (Hamamatsu Photonics, Japan). After filtering at 20 or 50 Hz the fluorescence ratio 410/470 was delivered on-line by an analog divider (Burr Brown DIV100). The background fluorescence was subtracted electronically in cell-attached mode. For [Ca2+]i off-line evaluation, the intracellular calibration procedure was used, described in detail before (Ganitkevich & Isenberg, 1991). The results are presented as both fluorescence ratio (410/470) and calibrated [Ca2+]i since the procedure can still be the subject of some errors (for example it does not allow for the possibility of cytoplasmic [Ca2J]i inhomogeneity during the transient). The myocytes were continuously superfused with physiological salt solution (PSS) composed of (mM): 150 NaCl, 3-6 CaCl2, 1-2 MgC12, 54 KCl, 20 glucose, 5 HEPES, adjusted with NaOH to pH 74. The pipettes were filled with an intracellular solution containing (mM): 130 KCl, 2 Na2ATP, 3 MgCl2, 10 HEPES, 0-1 K51ndo-1, adjusted with NaOH to pH 7-2. For a fast application of caffeine, a four-barrel glass pipette (0-1 mm openings) was placed at a distance about 0-25 mm from the cell from which solutions were pressure-applied. Application of Indo-1 containing PSS indicated that the solution change was complete within 0-2 s. One barrel contained PSS, thus the drugs could be rapidly washed out. The fast stream of solution as well as the contraction of the cell could potentially produce movement artifacts in the Indo-1 signal. Therefore, before each experiment a fast caffeine application (or application of PSS) was tested and cells with movement artifacts were excluded. All experiments were performed at 36 'C. The temperature was controlled by a heater which covered the tubing through which the solution flowed into the chamber. The solution in the application pipette was not heated: however. it was equilibrated with the solution in the chamber during the experiment since (i) the application pipette was usually up to 3 mm deep in the experimental chamber; (ii) the applied solution volume was not more than 30,ul even with long applications; (iii) applications of PSS produced no effect on membrane currents, which was expected in the case of the temperature differences. The results are expressed as means + S.D. of the mean. RESULTS

Changes in [Ca2+]i during wash-in and wash-out of caffeine Figure 1 shows the effect of a fast application of 10 mm caffeine on the fluorescence of Indo-1 (410 nm, upper trace; 470 nm, middle trace; fluorescence ratio 410/470, lower trace). At a holding membrane potential of -60 mV, caffeine was rapidly washed in and washed out from the solution surrounding the cell. This caffeine application resulted in a caffeine-induced [Ca2+]i transient. From the resting 120 nm, [Ca2+]i rose at a fast rate, a peak of 1250 nm being achieved within about 400 ms. During wash-out of caffeine [Ca2+]i decayed to 60 nm which was below the control resting [Ca2+]i (120 nM). In the context of this study, the decrease of [Ca2+]i to subresting levels is called 'undershoot of [Ca21]i '. Results similar to the one of Fig. 1 were obtained in a total of eighteen experiments. On average, at -60 mV following

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V. YA. GANITKEVICH AND G. ISENBERG

the caffeine treatment, [Ca2+]i fell to 47+12 nm, a value being 66+20 nm below resting (n = 18). Caffeine was shown to bind to Indo-1, quenching its fluorescence (O'Neill, Donoso & Eisner, 1990). During a 10 s exposure to caffeine, this effect was seen as the increase

F410! 4*

F470 Caffeine

0-28Ratio 41 0/470

Caffeine

[Ca2+]i (nM) 1150

4s Fig. 1. The effect of short application of caffeine on the fluorescence signals recorded from Indo-l loaded cell. Indo-l fluorescence at 410 nm (upper trace) and 470 nm (middle trace) is shown together with the fluorescence ratio (bottom trace, left ordinate) and computed [Ca2+], (bottom, right ordinate). At a holding potential of -60 mV, 1O mm caffeine was applied for 1 s (as indicated above the bottom trace). During the wash-out of caffeine, the [Ca2+], fell below the resting level. Simultaneously, the fluorescence signal at 470 nm was increased above the control value (indicated by *). Five depolarizing steps were applied (160 ms to 0 mV at 1 Hz) to activate Ca2+ influx through L-type Ca2+ channels for reloading the caffeine-sensitive store with Ca2 , then caffeine was applied a second time. Note, caffeine quenched the fluorescence of Indo-1.

of the noise of the fluorescence ratio (for example, Fig. 2). Although quenching is wavelength independent (O'Neill et al. 1990), the possibility of artifacts has to be excluded. During the wash-out of caffeine (Fig. 1) the increase in 470 nm fluorescence signal resulted from two processes; the dissociation of caffeine from Indo-1, shown to be proportional to the decay of the intracellular caffeine concentration (O'Neill et al. 1990), and the return of Ca2+-Indo-1 into the calcium-free form of Indo-1. When the fluorescence ratio revealed the [Ca2+]i undershoot, the 470 nm fluorescence signal increased beyond control (indicated by *). Although we cannot dissociate the two events, Fig. 1 clearly shows that the appearance of the [Ca2+]i undershoot goes along with an increase in the 470 nm fluorescence beyond control. Thus, quenching of Indo-1 by caffeine cannot explain the [Ca2+]i undershoot. The experiments were performed at a holding potential of -60 mV which prevented Ca2+ influx through L-type Ca2+ channels. At this potential the sensitivity of Ca2+-activated K+ channels to [Ca2+]i is low. Nevertheless the caffeine-induced [Ca2+]i transient was accompanied by outward Ca2+-activated K+ currents (IK, Ca) the amplitude of which varied from cell to cell (for example Figs 2, 3B, 4A and 5A). Within 1 min at -60 mV, [Ca2+]i had recovered to resting level following the short

103 CAFFEINE-SENSITIVECa2+ STORE IN SMOOTH MUSCLE caffeine treatment in about 20% of the cells; an example is given in Fig. 2. The recovery of [Ca2+]i from undershoot to the resting value could be facilitated by a train of five 160-ms-long voltage-clamp pulses to 0 mV. The pulses activated Ca2+ influx through L-type Ca2+ channels, part of the inflowing Ca2+ was sequestered into the intracellular stores, filling them (see below). Caffeine

0-271 Ratio 410/470

Caffeine

[Ca2] i(nM) 1260

-520

0.12J

-120 1 nA

8s

Fig. 2. The undershoot of [Ca2+]i after a 1 s and a 10 s caffeine application. Between the caffeine applications a train of five depolarization pulses (160 ms from -60 to 0 mV at 1 Hz) was applied. Upper trace, ratio of Indo-1 fluorescence (410/470, left hand scale) and calibrated [Ca2+], (right hand scale). Bottom trace, whole-cell net membrane current. The duration of caffeine application is indicated above the traces.

Figure 2 shows a first caffeine-induced [Ca2+]i transient with a peak to 1260 nm and a [Ca2+]i transient in response to a second caffeine application which peaked to only 520 nm. On average, the peak of the first caffeine-induced [Ca2+]i transient was 1490 + 332 nm and the peak of the second was 660 + 204 nm (for interval between caffeine applications between 4 and 8 s, n = 5), being, thus, 44 % of the first one. The differences between the second and the following responses to caffeine were less pronounced (Figs. 3B and 4A). However, within about 10 min the caffeine-induced [Ca2+]i transients exhibited 'run-down', i.e. they showed a slow rate of rise and a low peak. It is possible that cell dialysis may remove some intracellular constituents that are essential for Ca2+ sequestration and/or Ca2+ release. Following the wash-out of caffeine the undershoot of [Ca2+]i was recorded in all cells tested. The [Ca2+]i during the undershoot and the rate of recovery towards the resting level, however, were subject to variability. In the experiment shown in Fig. 2 [Ca2+]i had recovered close to control resting [Ca2+]i within about 30 s whereas the peak of the second [Ca2+]i transient remained attenuated. Upon a second wash-out of caffeine [Ca2+]i fell to 50 nm, i.e. to a lower level than during the first [Ca2+]i undershoot (65 nM, Fig. 2). Obviously, the degree of subresting [Ca2+]i did not increase accordingly to the peak of the preceding caffeine-induced [Ca2+]i transient, in contrast to results of Becker, Singer, Walsh & Fay (1989).

Caffeine depletes the stores of releasable Ca2+ Figure 3 shows [Ca2+]i transients in response to long exposures to caffeine (10 s in A, 25 s in B). In the continuous presence of caffeine [Ca2+]i fell at a rate that was lower than after wash-out of caffeine (compare Fig. 3B, second and third applications, where the decay of [Ca2+]i started from the same peak). In the presence of caffeine, [Ca2+]i fell from the peak, approaching slowly the control resting value. The

14V. YA. GANITKEVICH AND G. ISENBERG

104

undershoot of [Ca2+]i was never observed in the presence of caffeine. In none of the experiments with a long caffeine exposure did [Ca2+]i recover from the undershoot at a holding membrane potential of -60 mV. However, recovery could be achieved by a train of depolarizing pulses (see later).

A

B

Caffeine 0.34 -1600

[a2

,(m

F Ratio

[Ca 2] (n m)

Cafein

0-35i-

p1790

Ratio l\F700

]7|150 410/470 0/470410-b7L

Ratio

41

35

0-12

~4_

100

1°5 nA l_____ 8s

05 nA

2 6s

C

is Fig. 3. Caffeine deprived the store of releasable Ca2+. A, upper trace [Ca2+]j; lower trace, membrane current at -60 mV. A 10 s caffeine application was followed by two short applications (1 s each). [Ca2+]i decayed in the presence of 10 mm caffeine to the resting 95 nm and during wash-out of caffeine to 35 nm. The second (2-5 s after wash) and third caffeine application (13 s after wash) induced [Ca2+]i transients with a low rate of rise and small amplitude, as if the store contained a reduced amount of releasable Ca2+. B, upper trace, [Ca2+]i; lower trace, membrane current at -60 mV. The first caffeine application induced [Ca2+]i transient which peaked from 100 to 1790 nm and decayed to 60 nM. Simultaneously, an outward Ca2+-activated K+ current was recorded. The second [Ca2+]i transient (6 s after wash) peaked only to 700 nm. The third caffeine application (10 s after wash of second application) lasted for 26 s, long enough for the decay of [Ca2+]i to the resting 100 nM. Note that in the presence of caffeine quenching the Indo-1 fluorescence the noise of the ratio increased. C, the [Ca2+]i and membrane current during the first caffeine application shown in panel B are superimposed at an expanded time scale. Note the dissociation of the time course of [Ca2+]i and of IK Ca

The deprivation of the caffeine-sensitive Ca21 store of releasable Ca2+ was tested by subsequent caffeine applications. After a long caffeine exposure, the stores were deprived of releasable Ca2+ to an extent larger than after the short caffeine application (see Fig. 3); the rate of rise and the peak of the caffeine-induced [Ca2+]i transients were strongly diminished after long caffeine treatment (Figs 3A and 4B). This conclusion was further supported by the result that the rapid increase of [caffeine]o from 10 to 30 mm increased [Ca2+]i only slowly and from 130 to 270 nM (Fig. 8A), although 30 mm caffeine is expected to activate the Ca2+ release channels to a large extent and, partially, in a [Ca2+]i-independent way (Sitsapesan & Williams, 1990). Thus, the results suggested that the long exposure to caffeine had strongly deprived the kSR of releasable Ca2 .

CAFFEINE-SENSITIVE Ca2+ STORE IN SMOOTH MUSCLE

105

The first caffeine-induced [Ca2+]i transient in Fig. 3B which peaked to 1790 nm was associated with a prominent IK Ca' while no change in the current was resolved during the following caffeine treatment, when [Ca2+]i reached the peak 700 nm. The result can be explained by the low Ca2+-sensitivity of Ca2+-activated K+ channels at -60 mV. More importantly, the time course of the [Ca2+]i transient and IK, Ca were always dissociated (Fig. 3 C). The peak of IK, Ca occurred usually 200 to 400 ms earlier than the peak of [Ca2+]i. Also, IK Ca fell from the peak before the [Ca2+]i indicated by Indo- 1 started to decrease (Fig. 3 C). Since IK, Ca reflects the Ca2+ concentration close to the inner side of the membrane the comparison suggests that this local Ca2+ concentration can differ from the 'global' [Ca2+]i indicated by the Indo- 1 fluorescence.

Ca21 influx with ICa restores the load of caffeine-sensitive Ca2+ store After a short caffeine-induced [Ca2+]i transient when [Ca2+]i fell to the undershoot the following three caffeine applications induced [Ca2+]i transients of progressively smaller amplitude, i.e. [Ca2+]i reached peaks of 1260, 365, 290 and 290 nm during first, second, third and fourth caffeine treatment, correspondingly (Fig. 4A). Thus, a caffeine-sensitive Ca2+ store was partly deprived of releasable Ca2+ following caffeine treatment. After the fourth caffeine-induced [Ca2+]i transient five 160 ms pulses from the holding potential -60 to -0 mV were applied, to induce Ca2+ influx with ICa (Fig. 4A). This resulted in considerable recovery of the Ca2+ load of caffeine-sensitive store as was indicated by the following caffeine treatment which now induced a [Ca2+]i transient which peaked to 670 nM (Fig. 4A). A long (10 s) caffeine application deprived the caffeine-sensitive Ca 2+ store to such an extent that the following caffeine application produced only a small [Ca2+]i response (Fig. 4B). Following a train of 160 ms depolarizing pulses from -60 to 0 mV, evoking ICa, the load of the store was greatly recovered, i.e. the caffeine application now induced a [Ca2+]i transient which peaked to 430 nm compared with 90 nm before cell Ca2+ load with IC. The results of Fig. 4 have shown that Ca2+ influx through L-type Ca2+ channels ('Ca) can reload the caffeine-sensitive store. Whereas this hypothesis is well accepted, there is a discussion about other pathways of reloading (Putney, 1990; Missiaen, Wuytack, Raeymaekers, De Smedt, Droogmans, Declerck & Casteels, 1991; Bourreau, Abela, Kwan & Daniel, 1991). Most importantly, this pathway has been suggested to be under the control of the SR Ca2+ load (Missiaen, Declerck, Droogmans, Plessers, De Smedt, Raeymaekers & Casteels, 1990; Putney, 1990). In a recent study of smooth muscle cells from the rabbit jejunum Pacaud & Bolton (1991) suggested that Ca2+ deprivation of the store by caffeine activates Ca2` influx through voltage-independent channels. [Ca2+]i was increased at the membrane potentials negative to -40 mV where Ca2+ influx through L-type channels was without importance; this increase was accelerated at more negative membrane potentials. We examined, therefore, the effect of membrane potential on the [Ca21] during undershoot following caffeine treatment. In the experiment shown in Fig. 5A, during the period of the undershoot the membrane potential was stepped from -60 to - 100 mV for 8 s. There was no effect of membrane hyperp44rization on [Ca2+]i (Fig. 5A). When the membrane was held

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V. YA. GANITKEVICH AND G. ISENBERG

at -100 mV and a step to -60 mV was imposed, [Ca2+]i did not change either (not shown). Negative results for the influence of membrane potential on [Ca2+]i between -60 and -100 mV were obtained in all six cells tested. The possibility of the influence of membrane potential on Ca2+ influx refilling the stores was studied also with ramp-like membrane depolarizations applied during the

A

Caffeine

---

-

[Ca2+], (nM)

-

0 27RatioI

1260

|

410/4701

L330 0.1-1_----90 40

iK

1 nA

4s B

Caffeine

-

[Ca2+]j (nM) 1920

0*37-

Ratio

410/47043 012 J

90

-

1 nA

4s

Fig. 4. Ca2+ influx through depolarization-activated L-type Ca2+ channels reloads the caffeine-sensitive store with releasable Ca2+. Upper traces in A and B, [Ca2+],; lower traces, membrane currents. The caffeine was applied at times indicated above traces in A and B. A, during four repetitive caffeine applications (1 s duration, at 025 Hz), peak [Ca2+]i reached and the rate of the caffeine-induced [Ca2+]1 changes were progressively depressed. The train of five depolarizations (160 ms pulses from -60 to 0 mV at 1 Hz), greatly recovered both peak and the time course of caffeine-induced [Ca2 ]i transient. B, a 10 s caffeine application largely depleted the store, as indicated by the small response to the second caffeine application. A train of depolarizing pulses producing Ca2+ influx with ICa (160 ms from -60 to 0 mV at 1 Hz) reloaded the store with Ca2+ since the response to the third caffeine application partially recovered.

period of [Ca2+]i undershoot. Between -100 and -35 mV [Ca2+]i did not change (Fig.. 5B). An increase in [Ca2+]i was observed only when the membrane potential became positive to -35 mV (n = 4). This result is compatible with the hypothesis that Ca21 influx through L-type Ca2+ channels is the main pathway for reloading the cellular Ca2+ stores with releasable Ca2", at least in this type of smooth muscle cell.

CAFFEINE-SENSITIVE Ca2+ STORE IN SMOOTH MUSCLE

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Suppression of plasmalemmal Ca2+ fluxes with La3+ Lanthanum blocks both ICa and Ca2+ extrusion via the plasmalemmal Ca2+ATPase (PMCa, Kwan, Takemura, Obie, Thastrup & Putney, 1990). Unfortunately, the solution containing 10 mm caffeine and 3 mm La3+ precipitated. Therefore, the [Ca [] (nM) 2010

Caffeine 0-36

B

[Ca2,] i(nM)

Caffeine

[ 0.28 Ratioi1I

041RO/4702_~

1 410

:03 ,,---|-120

r1440

------O-3150

i

0 5 nA

0 mV

8s -60 mV

-60 mV -100 mV

-100 mV

Fig. 5. Effect of the membrane potential on [Ca2+], during the undershoot. A, upper trace, [Ca2+],; middle trace, membrane current; lower trace, voltage protocol. After caffeine application, [Ca2+], fell to the subresting level and the membrane potential was stepped from -60 to -100 mV for 8 s. B, upper trace, [Ca2+]1; lower trace, voltage protocol. After caffeine application at -60 mV, the membrane was hyperpolarized to -100 and depolarized at a constant rate (20 mV s-1). Note that subresting [Ca2+]i did not change until the membrane potential had become more positive than -35 mV.

caffeine-containing solution applied to the cell was La3+-free, and with the wash-out of caffeine, La3+ was reapplied. When caffeine was applied to the cell pretreated with 3 mm La3+ for approximately 10 s it induced a [Ca21]i transient that reached a usual peak and following wash-out of the caffeine decayed to the resting [Ca2+]i (Fig. 6A). In the presence of La3+, the undershoot of [Ca2+]i did not occur (n = 4). In the continuous presence of La3+, the second caffeine application induced a [Ca2+]i transient that reached the same peak as during the first one (Fig. 6A). These results favour the hypothesis that undershoot of [Ca2+]i and depletion of releasable Ca2+ are linked to a La3+-sensitive Ca2+ extrusion via the PMCa. The effect of the short caffeine application (Fig. 6A) is consistent with the idea that during the wash-out of La3+ some of it remained bound and blocked the PMCa. During the following 6 s caffeine application there was a slow decay of [Ca2+]i in the continuous presence of caffeine which was, however, much slower than in experiments without La3+ pretreatment (compare to Figs 3A and B, 4B). Due to the above problems it cannot be excluded that La3+ was partially washed out during long caffeine application with the result that PMCa could reduce [Ca2+]i. Following washout of caffeine [Ca2+]i approached the resting level (Fig. 6A).

V. YA. GANITKEVICH AND G. ISENBERG

108

The [Ca2+]i transients induced by a short (1 s) caffeine application in the absence and presence of La3+ are compared in Fig. 6B. Following the wash-out of caffeine, the caffeine-induced [Ca2+]i transients decayed along a similar time course in the absence and in the presence of La3+ (Fig. 6B). These results supported the hypothesis that it

A Caffeine La 3+

La3+

0 33Ratio 410/470

_____

________________

[Ca 2+]] (nM)

1800 I [70

I

0-12J

-7

1 nA

6s B

Caffeine

La3+

[Ca ]j (nM) .-1790

0 35-

[170

Ratio

410/4701.15

1

nAl t

_

I l

I I

|

6s Fig. 6. Effect of La3+ (3 mm) on depolarization- and caffeine-induced [Ca2+]1 transients. Upper traces in A and B, [Ca2+]i; lower traces, membrane currents. Application of La3+ and caffeine is indicated above traces in A and B. Note, since the mixture of 3 mm LaCl3 and 10 mm caffeine precipitated, the solutions contained either La3+ or caffeine. Thus, La3+ was washed out during the caffeine application, and during wash-out of caffeine La3+ was reapplied. A, [Ca2+]i transients induced by 2 s and 6 s caffeine applications after pretreatment with La3+. B, comparison of the caffeine-induced [Ca2+], transients before (left) and after application of 3 mm La3+. Note, La3+ was applied during the decay of the depolarization-induced [Ca2+]i transient. Following depolarizations did not evoke [Ca2+]i transients since Ca2+ currents were blocked by La3+.

is the SR Ca2+ pump that determines the decay of the caffeine-induced [Ca2+]i transient (in the absence of caffeine, see Discussion). This idea is further supported by the result that the decay of the depolarization-induced [Ca2+]i transient was not retarded by La3+ applied immediately after the peak of the depolarization-induced [Ca2+]i transient (Fig. 6B).

CAFFEINE-SENSITIVE ,a2+ STORE IN SMOOTH MUJSCLE

109

Depolarization-induced [Ca21]i transients during the undershoot of [Ca2+]i Since membrane depolarization induces 'Ca and concomitant Ca2+ release from caffeine-sensitive Ca2+ stores (Ganitkevich & Isenberg, 1992b), depolarizationinduced [Ca2+]i transients can be used to study the modulation of cellular Ca2+

0.32-1

Caffeine l

[Ca2+]i (nM) 1600

Ratio

-470 110

410/470